Fluorescence labeling is a particularly useful tool for marking a protein or cell of interest. Traditionally, a protein of interest is purified and then covalently conjugated to a fluorophore derivative. For in vivo studies, the protein-dye complex is then inserted into the cells of interest using micropipetting or a method of reversible permeabilization. The dye attachment and insertion steps, however, make the process laborious and difficult to control.
Another way of labeling a protein of interest is to concatenate the gene expressing the protein of interest and a gene expressing a marker, and then express the fusion product. Typical markers include .beta.-galactosidase, firefly luciferase and bacterial luciferase. These markers, however, require exogenous substrates or cofactors and are therefore of limited use for in vivo studies.
A marker that does not require any exogenous cofactor or substrate is the green fluorescent protein (GFP) of the jellyfish Aequorea victoria, a protein with an excitation maximum at 395 nm and an emission maximum at 510 nm. Uses of GFP for the study of gene expression and protein localization are discussed in more detail in papers by Chalfie et al. in Science 263 (1994), p. 802-805, and Heim et al. in Proc. Nat. Acad. Sci. 91 (1994), p. 12501-12504. Some properties of wild-type GFP are disclosed for example in papers by Morise et al. in Biochemistry 13 (1974), p. 2656-2662, and Ward et al. in Photochem. Photobiol. 31 (1980), p. 611-615. An article by Rizzuto et al. in Curr. Biology 5 (1995), p. 635-642 discusses the use of wild-type GFP as a tool for visualizing subcellular organelles in cells, while a paper by Kaether and Gerdes in Febs Letters 369 (1995), p. 267-271, reports the visualization of protein transport along the secretory pathway using wild-type GFP. The expression of GFP in plant cells is discussed in an article by Hu and Cheng in Febs Letters 369 (1995), p. 331-334, while GFP expression in Drosophila embryos is described in a paper by Davis et al. in Dev. Biology 170 (1995), p. 726-729.
GFP is a 238-amino acid protein, with amino-acids 65-67 involved in the formation of the chromophore. A biosynthetic scheme for the chromophore is proposed in the above-mentioned article by Heim et al. (1994), and is shown in FIG. 1. Some of the newly translated protein precipitates as non-fluorescent inclusion bodies. For the protein that does not precipitate, amino acids 65-67 may be involved in cyclization and oxidation to form the chromophore. The time constant for chromophore formation was observed (Heim et al., 1995) to be on the order of two hours, suggesting that wild-type GFP would not be a practical marker for monitoring fast changes in gene expression.
Wild-type GFP has a major excitation peak at 395 nm and a minor excitation peak at 470 nm. The absorption peak at 470 nm allows the monitoring of GFP levels using standard fluorescein isothiocyanate (FITC) filter sets and the 488 nm line of an Ar ion laser. The ability to excite GFP at 488 nm also permits the use of GFP with standard fluorescence activated cell sorting (FACS) equipment. The emission levels of wild-type GFP excited at 488 nm are relatively low, however, and the resulting low signal-to-noise ratio and limited dynamic range limit the use of GFP with typical FACS equipment. Mutations in GFP leading to brighter emission following 488 nm excitation would be of value in many applications, including FACS.
Mutations in GFP which shift the excitation maximum from 395 nm to about 490 nm have been reported by Delagrave et al. in Biotechnology 13 (1995), p. 151-154, and Heim et al. in Nature 373 (1995), p. 663-664. The above-mentioned articles by Delagrave et al. and Heim et al. (1995) are herein incorporated by reference. Mutants with Ala, Gly, Ile, Cys or Thr substituted for Ser65 had large shifts in excitation maxima, and fluoresced more intensely than wild-type protein when excited at 488 nm. Other mutants with altered spectra are disclosed in the previously-mentioned paper by Heim et al. (1994). A summary of the characteristics of the mutants disclosed in the two Heim et al. papers (1994 and 1995) is given in Table 1:
TABLE 1 ______________________________________ Excitation Emission Relative Mutation maximum maximum fluorescence (%) ______________________________________ None 396 nm 508 nm =100 Ser-202 to Phe 398 nm 511 nm 117 (w/ 395 nm exc) Thr-203 to Ile Ile-167 to Val 471 nm 502 nm 166 (w/ 475 nm exc) Ile-167 to Thr 471 nm 502 nm 188 (w/ 475 nm exc) Tyr-66 to His 382 nm 448 nm 57 (w/ 395 nm exc) Tyr-66 to Trp 458 nm 480 nm Not done Ser-65 to Thr 489 nm 511 nm .about.600 Ser-65 to Cys 479 nm 507 nm .about.600 ______________________________________
The mutation of Ser65 to Thr or Cys was observed to increase by a factor of 6 the fluorescence of GFP following 488 nm excitation. However, further improvement in the fluorescence intensity of GFP excited at 488 nm would clearly be desirable.